Gel electrophoresis is a common procedure for the separation of biological molecules, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polypeptides and proteins. In gel electrophoresis, the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.
The basic apparatus used in this technique consists of a gel often enclosed in a glass tube or sandwiched as a slab between glass or plastic plates. The gel has an open molecular network structure, defining pores, which are saturated with an electrically conductive buffered solution of salts. These pores through the gel are large enough to admit passage of the migrating macromolecules.
The gel is placed in a chamber in contact with buffer solutions which make electrical contact between the gel and the cathode and anode of an electrical power supply. A sample containing the macromolecules and a tracking dye is placed on top of the gel. An electric potential is applied to the gel causing the sample macromolecules and tracking dye to migrate toward the bottom of the gel. The electrophoresis is halted just before the tracking dye reaches the end of the gel. The locations of the bands of separated macromolecules are then determined. By comparing the distance moved by particular bands in comparison to the tracking dye and macromolecules of known mobility, the mobility of other macromolecules can be determined. The size of the macromolecule can then be calculated or macromolecules of different sizes can be separated in the gel.
There are a wide range of gel-forming materials used for electrophoresis. Polyacrylamides, polymethacrylamides and other related polymers are preferred for separation of smaller molecular weight materials such as proteins, peptides and small nucleic acids. Conversely, agarose, cellulose acetate and starch are preferred for larger molecules. These gel materials are typically compatible with aqueous systems, though some are also compatible with non-aqueous solvents.
Formation of the gel material to the desired physical dimensions can be accomplished by varying techniques, depending on the material chosen. With agarose or gelatin the common method is to heat the polymer causing the material to go into solution. The solution can then be poured into a cast and allowed to polymerize by cooling. Alternatively, polyacrylamides, polymethacrylamides and other related polymers can be chemically polymerized by various means including free radical induced polymerization with ammonium persulfate and tetramethylethylenediamine.
A common problem with gel electrophoresis is the difficulty of removing the protein, nucleic acid or other analyte of interest from the gel after it has been separated from other components. Because the gel matrix has very small pore sizes, large molecules do not easily diffuse out of the gel matrix after they have been drawn into the matrix through electromotive force. Proteins also do not diffuse into the gel matrix readily in the absence of electromotive force. The larger the molecular weight of the protein the more difficult it is to get the molecules into or out of the gel. Thus, techniques have been developed for preparing a gel for extraction or introduction of molecules out of or into the gel.
The state of the art includes a procedure for gel subdivision using a sieve. Christoph Eckerskom and Rudolf Grimm describe the use of a stainless steel sieve placed in the end of a syringe barrel for subdividing gels. Eckerskorn and Grimm attribute their technique to J. Heukeshoven and R. Dernick, as described in B. Radola (ed.), Electrophoresis Forum xe2x80x291, Technical University, Munich 1991, pp. 501-506. In another article, J. Lila Castellanos-Serra, et al., Electrophoresis 1999; 20: 732-737, a stainless steel sieve screen is used for subdividing gels to remove proteins for further analysis. This article attributes the idea to Eckerskorn and Grimm and to Heukeshoven and Dernick. Castellanos-Serra et al. placed a piece of stainless sieve screen in the narrow end of a syringe barrel and used the pressure of the plunger to force the gel through the screen.
However, this approach has significant disadvantages. First, the syringe is costly, especially if there are large numbers of gels to be processed simultaneously. This cost arises in part because the syringe comprises several parts including a barrel, plunger and gasket. Second, the user must manually force the gel through the mesh with the action of a plunger. This technique is significantly labor intensive and is not amenable to automation. Third, the plunger does not advance all of the gel cleanly through the mesh, because the force applied by the plunger stops at the top of the mesh.
Others have demonstrated that centrifugal force can be useful in forcing materials through a barrier for various purposes, e.g. a filtration membrane for separation. For example, U.S. Pat. No. 3,583,627 to Wilson describes concentrating a large molecular weight substance in solution by fixing a filter into the end of the upper of two nested tubes and spinning the tubes to force the solvent through the filter while retaining the macromolecules. There are numerous examples of using this basic principle to concentrate macromolecules, for example U.S. Pat. No. 4,632,761 Bower et al. These devices and methods, however, are designed for filtration of solutions and are not suitable for cutting or subdividing a semi-solid gel or other substance.
Additionally, Millipore Corporation of Bedford, Mass. currently sells Product No. 42600 xe2x80x9cUltrafree DA for DNA extraction,xe2x80x9d for the subdivision of gel fragments. This product also suffers from several drawbacks. First, it uses a nested tube set, the upper tube having in its base plastic projections molded in place that are supposed to subdivide a gel when the tube set is spun. However, in practice, these do not work well because the resulting subdivided gel has large, inconsistently-sized pieces of gel leading to inefficient and unreliable extraction. Second, the Millipore device is recommended primarily for agarose gels and may be used on polyacrylamide gels, but only with a maximum polymer concentration of 10% by weight. Gels having a polymer concentration less than 10% by weight are usually unable to efficiently separate very low molecular weight peptides and proteins, which often require 12%, 15%, 18% or a higher percentage of polymer concentration by weight.
There is, therefore, a need for a method of easily, efficiently, reliably and inexpensively subdividing a gel to facilitate the extraction of various molecules from the gel, or conversely, the introduction of molecules into the gel. In particular, as more and more analytes become available for study, e.g. through the Human Genome Project and follow-on projects to identify genes and express gene products, there is a need to perform such extractions or introductions in high-throughput and automated formats.
Although much of the description of the invention is related to removing proteins from gels, it is also often of interest to remove other molecules from gels. A person of ordinary skill in the art would readily apply the techniques described herein to other molecules commonly subjected to electrophoresis, e.g. nucleic acids such as DNA or RNA.
The present invention provides a convenient method of subdividing a gel containing a protein, nucleic acid or other analyte of interest into small, consistently-sized fragments, which facilitate the diffusion of reagents into, or analyte out of, the gel. In one embodiment, the apparatus of the present invention consists of a centrifuge tube incorporating a mesh or grid barrier, through which the gel is drawn by centrifugal force when the tube is spun, thereby forming a mincing tube. The mesh or grid is preferably one having small and consistent hole spacing within and between different manufacturing lots.
The mesh material extends across the end of the tube. To subdivide a gel using the mincing tube, a gel is placed upon the mesh material and the tube. When the mesh material and the gel are spun in a centrifuge, the gel is drawn through the mesh material so that the gel is subdivided into generally uniform smaller fragments.
The mesh material may be secured to a tube in the form of a nesting tube. The nesting tube nests within the opening of a recovery vessel. Alternatively, the mesh material may be placed in series with a conditionally porous membrane in the nesting tube. Centrifuging the nesting tube and the recovery vessel subdivides gel material into fragments by forcing the gel through the mesh material. The gel subsequently falls upon the membrane, and may be treated on the membrane to extract or otherwise treat analytes in the gel material.
In an alternate embodiment, the centrifuge tube may comprise parts or segments, with one part of the nested set including the mesh material to subdivide the gel. Another part provides a porous membrane or a conditionally porous membrane. Another part provides reversed phase capture material, either held in place by a membrane or by using membrane derived so as to bind proteins by hydrophobic interactions. Another part provides the immobilized antibody to capture a high abundance protein. Several types of such immobilized antibodies might be provided either as separate segments for nesting or combined in a single segment. The last segment can be a receptacle or recovery vessel for fluid driven through the column by centrifugal force.
A particular advantage of this invention is that it makes the process of subdividing the gel simple and suitable for automation. It is often the case that numerous samples must be processed for further analysis, such as determining their amino acid sequence. The device and method of the present invention avoids significant hands-on work, such as using a spatula to chop-up or crush a gel, prior to the extraction process.
One advantage of the present invention is that it significantly decreases the time required to elute a protein, or other analyte, from the gel or to get homogeneous distribution of a reagent being diffused into the gel. This advantage is achieved because the invention results in a very finely subdivided gel. Because the protein or other analyte must diffuse out of the gel matrix, the farther it must diffuse, the longer it takes to extract. Therefore, a very finely subdivided gel allows proteins, for example, to be quickly diffused into or out of a gel.
Further, the present invention results in consistent thickness of the gel fragments, which allows the diffusion distance for reagents going into the gel or diffusing out of the gel to be consistent. The time required for penetration of the fragments of subdivided gel by reagent, or diffusion of materials out of the fragments, will be consistent and reproducible only if the sizes of the fragments are consistent and reproducible. Gels subdivided manually will not have this consistency and therefore the amount of protein, for example, which diffuses out of the gel will be variable leading to inconsistent results in further characterization. Often, samples of gel contain only tiny quantities of protein to be used for subsequent characterization, so consistency and efficiency of elution from the gel matrix is very important.
Thus, the method and device of the present invention offer several advantages. First, the syringe described by Eckerskorn et al. is far more costly than a centrifuge tube. Second, centrifuging methods, as described in the present invention, are more readily and cheaply adaptable to automation than methods employing syringe/plunger-and-mesh devices. Third, the use of centrifugal force on the small gel fragments draws the fragments completely through the sieve and down into the collecting tube, thereby solving the problem in the prior art of gel material remaining in the syringe mesh after the motive force of pressure is removed. In contrast to the Millipore product (Product No. 46200) with its molded projections for subdividing the gel, the sieve approach of the present invention results in smaller and more consistent gel fragments resulting in more efficient and reliable analyte extraction. The present invention also, unlike the Millipore product, subdivides gel with polyacrylamide concentrations greater than 10%, because the centrifugal force effectively drives the gel through the sieving mesh material.
In additional specific embodiments, methods and apparatuses of the invention are used to subdivide gels having polyacrylamide concentrations ranging from about 3% to 10%, about 5% to 10%, about 10% to 11%, about 10% to 12%, about 10% to 15%, about 10% to 18%, about 10% to 20%, about 12% to 15%, about 12% to 18%, about 12% to 20%, about 15% to 18%, about 15% to 20% and about 18% to 20%. Likewise, in other specific embodiments, the invention subdivides any other gel types, including, but not limited to, agarose gels.
In one aspect of the invention, a method for subdividing a semisolid material using a mincing tube having a mesh material disposed therein, comprises the steps of: placing the semisolid material upon the mesh material of the mincing tube; and centrifuging the mincing tube, the mesh, and the semisolid material to facilitate passage of the semisolid material through the mesh, thereby subdividing the semisolid material into fragments. Further steps may comprise: introducing an extraction solution into the mincing tube to extract an analyte from the semisolid material; incubating the mincing tube including the extraction solution and the semisolid material; and eluting the analyte from the subdivided semisolid material, wherein the extracted analyte may be a macromolecule, or alternatively, at least one or more of: proteins, peptides, nucleic acids and carbohydrates. Furthermore, the mesh material may span a lumen of the mincing tube, and the mesh material may be concave from a top edge of the mincing tube.
Further steps may include introducing an extraction solution into the mincing tube to extract analytes from the semisolid material, wherein the extraction solution and the analytes create an analyte solution; and transferring the analyte solution into a recovery vessel, the recovery vessel having a conditionally porous material disposed therein, such that the analyte solution may be in contact with the conditionally porous material. Additionally, the method may include centrifuging the recovery vessel with the analyte solution, such that some or all of the analyte solution flows through the conditionally porous material, wherein the conditionally porous material comprises one or more components selected from the group consisting of a long-chain alkyl group, an ion exchange group, a short chain carboxylate or sulfonate, an affinity group (e.g., an antibody), streptavidin, a chelating group or a boronic acid. In one aspect of the method, the conditionally porous material may be a polyvinylidene difluoride membrane a nylon membrane, a nitrocellulose membrane and/or a glass fiber membrane and the semisolid material may be an electrophoresis gel, or a subportion thereof.
In another aspect of the invention, a method for the treatment of a semisolid material using a first treating tube having a conditionally porous material disposed therein, comprises the steps of: combining the semisolid material with reactants in the first treating tube to create a reaction mixture; and centrifuging the first treating tube, such that some or all of the reaction mixture may be drawn through the conditionally porous material. The method may further comprise placing the first treating tube in a recovery vessel, such that the first treating tube may be nested into the recovery vessel; and capturing the reaction mixture in the recovery vessel. Alternatively, the method may further comprise: providing a second treating tube for nesting with the recovery vessel, wherein the second treating tube includes a second conditionally porous material; nesting the first treating tube in the second treating tube and nesting the second treating tube in the recovery vessel; centrifuging the first and the second treating tubes; and capturing the reaction mixture in the recovery vessel. The conditionally porous material may be a polyvinylidene difluoride membrane, a nylon membrane, a nitrocellulose and/or a glass fiber membrane and, in one aspect of the method, the first treating tube may be an array of treating tubes for aligning and mating with a microtiter plate. The first treating tube may include a mesh material at a first end of a lumen of the first treating tube, and the conditionally porous material may be disposed at a second end of the lumen of the first treating tube. The reactants may be disposed on the conditionally porous material, and the reactants may comprise one or more components selected from the groups consisting of a long-chain alkyl group, an ion exchange group, a short chain carboxylate or sulfonate, an affinity group (e.g., an antibody), streptavidin, a chelating group or a boronic acid. Furthermore, the semisolid material may be an electrophoresis gel, or a subportion thereof.
Another aspect of the invention includes a method for the division of a semisolid material using a mincing tube having a mesh material disposed therein, wherein the mincing tube may be nested in a recovery vessel such that substances passing through the mesh material are captured in the recovery vessel, the method comprising the steps of: placing a semisolid material in the mincing tube; and centrifuging the mincing tube and the recovery vessel until the semisolid material is divided into fragments by the mesh material. The method may further comprise the step of: providing a treating tube nested in series after the mincing tube and before the recovery vessel, wherein the treating tube includes a conditionally porous material disposed therein, and the conditionally porous material is in series with the mesh material. Further, the conditionally porous material may comprise one or more of the group consisting of: a long-chain alkyl group, an ion exchange group, a short chain carboxylate or sulfonate, an affinity group (e.g., an antibody), streptavidin, a chelating group or a boronic acid.
An apparatus for the subdivision of semisolid materials, may comprise: a mincing tube; and a mesh disposed in the mincing tube, wherein when the mincing tube is subjected to centrifugal forces, a semisolid material placed within the mincing tube on one side of the mesh is drawn through the mesh. The semisolid material may be an electrophoresis gel, or a subportion thereof, and may contain a protein or nucleic acid. Furthermore, the gel may have a polyacrylamide concentration greater than 10 percent or less than 10 percent. The mesh of the apparatus may be a metal or a polymeric mesh, such as a stainless steel mesh or a nylon mesh. The mesh may have a hole size ranging from 0.01 mm2 to 9 mm2, and the size of holes in the mesh may be substantially uniform. The mesh may cover an end of the mincing tube, and may be flat or formed to extend concavely into the mincing tube. The mesh may be fixed to the mincing tube by welding, by an adhesive, or by a compression ring.
The apparatus may be in a kit including a buffered solution (which may or may not comprise one or more extraction reagents such as one or more enzymes or the like), printed instructions for use of the apparatus, a spare mesh material, particles treated with, or having affixed thereto, an immobilized antibody, and a treating tube containing a conditionally porous mesh material disposed therein.
Another embodiment of the apparatus for the recovery of proteins and nucleic acids from a gel comprises: a mincing tube having a lumen, the mincing tube including a first conditionally porous material extending across the lumen and a mesh material extending across the lumen; and a recovery vessel disposed adjacent to the mincing tube, such that contents of the mincing tube flow through the mesh material and the conditionally porous material into the recovery vessel. The mincing tube may be nested within the recovery vessel. Additionally, the apparatus may include a treating tube containing a second conditionally porous material, wherein the treating tube is disposed adjacent the mincing tube so that the contents flow through the mesh material, the first conditionally porous material and the second conditionally porous material in series. The mincing tube may be nested within the treating tube, and the treating tube may be nested within the recovery vessel. In one aspect of the invention, the mincing tube and the treating tube are arrays of tubes that align with and mate with a microtiter plate. Contents of the apparatus may include an extraction buffer and proteins. Further, the first conditionally porous material may contain one or more of immobilized enzymes (e.g., proteases such as trypsin, chymotrypsin, pepsin, papain and the like), immobilized carbon chains and immobilized antibodies. The first conditionally porous material may be a polyvinylidene difluoride membrane, a nylon membrane, a nitrocellulose membrane and/or a glass fiber membrane. In one aspect, the mincing tube may comprise: a first portion containing the mesh; and a second portion containing the first conditionally porous material, such that the contents of the mincing tube flow through the mesh and the first conditionally porous material in series. The first portion may be nested with the second portion and the second portion may be nested with the recovery vessel. The first and the second portions may be arrays of tubes for aligning with and mating with a microtiter plate. The mesh material may be a metal or fabric mesh, wherein the mesh is a stainless steel mesh or a nylon mesh having a hole size ranging from 0.01 mm2 to 9 mm2.
Another embodiment of an apparatus for subdividing and processing a gel comprises: a mincing tube having a mesh material disposed therein; and a recovery vessel connected to the mincing tube, wherein a conditionally porous material is disposed within the recovery vessel. A reagent may be attached to the conditionally porous material, and the reagent may be one of immobilized trypsin, immobilized carbon chains and immobilized antibodies. The recovery vessel may be removably connected with the mincing tube in an inverted relationship. The mesh may a metal or fabric mesh, such as a stainless steel mesh or a nylon mesh having a hole size ranging from 0.01 mm2 to 9 mm2. The conditionally porous material may be a polyvinylidene difluoride membrane, a nylon membrane, a nitrocellulose membrane and/or a glass fiber membrane.